High affinity HIV T cell receptors

ABSTRACT

The present invention provides TCRs having high affinity. The TCR binds to SLYNTVATL (SEQ ID NO:16)-HLA-A*0201 with a K D  of less than or equal to 1 μM and/or an off-rate (k off ) of 1×10 −3  S −1  or slower using Surface Plasmon Resonance. The TCRs are non-native, isolated or recombinant. The TCRs are useful, either alone, or with a therapeutic agent, for targeting HIV infected cells that present the SLYNTVATL (SEQ ID NO:16)-HLA-A*0201 complex.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a Divisional of U.S. application Ser. No.11/887,536, filed Nov. 7, 2008, now U.S. Pat. No. 8,378,074 which is aNational Stage application of co-pending PCT applicationPCT/GB2006/001147 filed on Mar. 29, 2006, which was published in Englishunder PCT Article 21(2) on Oct. 5, 2006, and which claims the benefit ofGB 0506760.8 filed Apr. 1, 2005 and GB 0516487.6 filed Aug. 10, 2005.These applications are incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

The present invention relates to T-cell receptors (TCRs) having theproperty of binding to HIV Gag polypeptide-derived SLYNTVATL-HLA-A*0201.The TCRs comprise at least one TCR α chain variable domain and/or atleast one TCR β chain variable domain and have a K_(D) for the saidSLYNTVATL-HLA-A*0201 complex of less than or equal to 104 and/or has anoff-rate (k_(off)) for the SLYNTVATL-HLA-A*0201 complex of 1×10⁻³ S⁻¹ orslower.

BACKGROUND OF THE INVENTION

The Human Immuno-deficiency Virus (HIV) is the causative agent ofAcquired Immuno-deficiency Disease Syndrome (AIDS). The virus is anenveloped retrovirus belonging to the lentivirus group. The SLYNTVATL(SEQ ID NO: 16) peptide is derived from the g17 gene product of the Gaggene, one of nine genes which make up the Human Immuno-deficiencyVirus-1 (HIV-1) The peptide is loaded by HLA-A*0201 and presented on thesurface of HIV infected cells. Therefore, the SLYNTVATL-HLA-A2*0201complex provides an HIV marker that TCRs can target, for example for thepurpose of delivering cytotoxic or immuno-stimulatory agents to theinfected cells. However, for that purpose it would be desirable if theTCR had a high affinity and/or a slow off-rate for the peptide-HLAcomplex.

SUMMARY OF THE INVENTION

This invention makes available for the first time TCRs having anaffinity (K_(D)) of less than or equal to 104, and/or an off-rate(k_(off)) of 1×10⁻³ S⁻¹ or slower, for the SLYNTVATL-HLA-A*0201 complexPROVIDED THAT when the said TCR is presented by cell and comprises SEQID NOs: 1 and 2, the cell is not a native T cell. Such TCRs are useful,either alone or associated with a therapeutic agent, for targeting HIVinfected cells presenting that complex.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings.

FIGS. 1 a and 1 b detail the alpha chain variable domain amino acid andbeta chain variable domain amino acid sequences of the parental HIV GagTCR respectively.

FIGS. 2 a and 2 b show respectively the DNA sequence of soluble versionsof the parental HIV Gag TCR α and β chains.

FIGS. 3 a and 3 b show respectively the HIV Gag TCR α and β chainextracellular amino acid sequences produced from the DNA sequences ofFIGS. 2 a and 2 b.

FIGS. 4 a and 4 b show respectively the DNA sequence of soluble versionsof the HIV Gag TCR α and β chains mutated to enocode additional cysteineresidues to form a non-native disulfide bond. The mutated codon isindicated by shading and The introduced restriction enzyme recognitionsites are underlined.

FIGS. 5 a and 5 b show respectively the HIV Gag TCR α and β chainextracellular amino acid sequences produced from the DNA sequences ofFIGS. 4 a and 4 b. The introduced cysteine in each chain is indicated byshading.

FIGS. 6 a-6 c detail the alpha chain variable domain amino acidsequences of the high affinity HIV Gag TCR variants.

FIGS. 7 a and 7 b detail the beta chain variable domain amino acidsequences of the high affinity HIV Gag TCR variants.

FIG. 8 a details the amino acid sequence of a soluble portion of TRAC.

FIG. 8 b details the amino acid sequence of a soluble portion of TRBC1.

FIG. 8 c details the amino acid sequence of a soluble portion of TRBC2.

FIGS. 9 a and 9 b detail the DNA sequence of the pEX954 plasmid.

FIGS. 10 a and 10 b detail the DNA sequence of the pEX821 plasmid.

FIG. 11 details the beta chain amino acid sequences of the parentalsoluble HIV Gag TCR variant fused via a peptide linker to wild-typehuman IL-2. The amino acids of the linker and IL-2 are indicated initalics.

FIGS. 12 a and 12 b provide the Biacore response curves generated forthe interaction of the soluble disulfide-linked parental HIV Gag TCR andthe SLYNTVATL-HLA-A*0201 complex.

FIG. 13 provides a plasmid map of the pEX954 plasmid.

FIG. 14 provides a plasmid map of the pEX821 plasmid.

FIG. 15 a provides the full-length DNA sequence of the parental HIV GagTCR α chain optimised for expression in human T cells.

FIG. 15 b provides the full-length DNA sequence of the parental HIV GagTCR β chain optimised for expression in human T cells.

FIG. 16 a provides the full-length amino acid sequence of the parentalHIV Gag TCR α chain.

FIG. 16 b provides the full-length amino acid sequence of the parentalHIV Gag TCR β chain optimised for expression in human T cells.

FIG. 17 a provides FACS analysis data for untransduced controlCD8^(+ T cells.)

FIG. 17 b provides FACS analysis data demonstrating expression of theparental HIV Gag TCR on the surface of transduced CD8^(+ T cells.)

FIGS. 18 a and 18 b provide the amino acids sequences of the alpha andbeta chains of a soluble disulfide-linked high affinity c11c6 HIV GagTCR.respectively.

FIG. 19 a demonstrates the ability of soluble disulfide-linked highaffinity c11c6 HIV Gag TCRs to inhibit the activation of theSLYNTVATL-HLA-A*0201 reactive OX84 polyclonal T cell line in thepresence of To cells infected with HIV as measured by IFN-γ production.

FIG. 19 b demonstrates the ability of soluble disulfide-linked highaffinity c11c6 HIV Gag TCRs to inhibit the activation of theSLYNTVATL-HLA-A*0201 reactive OX84 polyclonal T cell line in thepresence of To cells infected with HIV as measured by TNF-α production.

FIG. 20 a demonstrates the ability of soluble disulfide-linked highaffinity c11c6 HIV Gag TCRs to inhibit the activation of theSLYNTVATL-HLA-A*0201 reactive OX84 polyclonal T cell line in thepresence of SLYNTVATL peptide-pulsed uninfected To cells as measured byIFN γ production.

FIG. 20 b demonstrates the ability of soluble disulfide-linked highaffinity c11c6 HIV Gag TCRs to inhibit the activation of theSLYNTVATL-HLA-A*0201 reactive OX84 polyclonal T cell line in thepresence of SLYNTVATL peptide-pulsed uninfected To cells as measured byTNF-α production.

FIG. 21 demonstrates the ability of soluble disulfide-linked highaffinity c11c6 HIV Gag TCRs to stain SLYNTVATL peptide-pulsed T2 cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a T-cell receptor (TCR) having theproperty of binding to SLYNTVATL-HLA-A*0201 and comprising at least oneTCR α chain variable domain and/or at least one TCR β chain variabledomain CHARACTERISED IN THAT said TCR has a K_(D) for the saidSLYNTVATL-HLA-A*0201 complex of less than or equal to 1 μM and/or has anoff-rate (k_(off)) for the SLYNTVATL-HLA-A*0201 complex of 1×10⁻³ S⁻¹ orslower, PROVIDED THAT when the said TCR is presented by cell andcomprises SEQ ID NOs: 1 and 2, the cell is not a native T cell.

The K_(D) and/or (k_(off)) measurement can be made by any of the knownmethods. A preferred method is the Surface Plasmon Resonance (Biacore)method of Example 4.

For comparison, the interaction of a disulfide-linked soluble variant ofthe parental HIV gag TCR (see SEQ ID NO: 9 for TCR α chain and SEQ IDNO: 10 for TCR β chain) and the SLYNTVATL-HLA-A*0201 complex has a K_(D)of approximately 85 nM and an off-rate (k_(off)) of 2.21×10⁻² S⁻¹ asmeasured by the Biacore-base method of Example 4.

The parental HIV Gag TCR specific for the SLYNTVATL-HLA-A*0201 complexhas the following Valpha chain and Vbeta chain gene usage:

Alpha chain—TRAV12.2

Beta chain:—TRBV 5.6

The parental HIV Gag TCR can be used as a template from which other TCRsof the invention with high affinity and/or a slow off-rate for theinteraction between said TCRs and the SLYNTVATL-HLA-A*0201 complex canbe produced. Thus the invention includes TCRs which are mutated relativeto the parental HIV Gag TCR α chain variable domain (see FIG. 1 a andSEQ ID No: 1) and/or β chain variable domain (see FIG. 1 b and SEQ IDNO: 2) in at least one complementarity determining region (CDR) and/orvariable domain framework region thereof. It is also contemplated thatother hypervariable regions in the variable domains of the TCRs of theinvention, such as the hypervariable 4 (HV4) regions, may be mutatedwithin a high affinity mutant TCR.

Phage display provides one means by which libraries of TCR variants canbe generated. Methods suitable for the phage display and subsequentscreening of libraries of TCR variants each containing a non-nativedisulfide interchain bond are detailed in (Li et al., (2005) NatureBiotech 23 (3): 349-354) and WO 2004/04404.

Native TCRs exist in heterodimeric αβ or γδ forms. However, recombinantTCRs consisting of a single TCR α or TCR β chain have previously beenshown to bind to peptide MHC molecules.

In one embodiment the TCR of the invention comprise both an α chainvariable domain and an TCR β chain variable domain.

As will be obvious to those skilled in the art the mutation(s) in theTCR α chain sequence and/or TCR β chain sequence may be one or more ofsubstitution(s), deletion(s) or insertion(s). These mutations can becarried out using any appropriate method including, but not limited to,those based on polymerase chain reaction (PCR), restriction enzyme-basedcloning, or ligation independent cloning (LIC) procedures. These methodsare detailed in many of the standard molecular biology texts. Forfurther details regarding polymerase chain reaction (PCR) mutagenesisand restriction enzyme-based cloning see (Sambrook & Russell, (2001)Molecular Cloning—A Laboratory Manual (3^(rd) Ed.) CSHL Press) Furtherinformation on LIC procedures can be found in (Rashtchian, (1995) CurrOpin Biotechnol 6 (1): 30-6)

It should be noted that any αβ TCR that comprises similar Valpha andVbeta gene usage and therefore amino acid sequence to that of the HIVGag TCR could make a convenient template TCR. It would then be possibleto introduce into the DNA encoding one or both of the variable domainsof the template αβ TCR the changes required to produce the mutated highaffinity TCRs of the invention. As will be obvious to those skilled inthe art, the necessary mutations could be introduced by a number ofmethods, for example site-directed mutagenesis.

The TCRs of the invention include those in which one or more of the TCRalpha chain variable domain amino acids corresponding to those listedbelow are mutated relative to the amino acid occurring at thesepositions in the sequence provided for the parental HIV Gag TCR alphachain variable domain in FIG. 1 a and SEQ ID No: 1.

Unless stated to the contrary, the TCR amino acid sequences herein aregenerally provided including an N-terminal methionine (Met or M)residue. As will be known to those skilled in the art this residue maybe removed during the production of recombinant proteins. As will alsobe obvious to those skilled in the art, it may be possible to truncatethe sequences provided at the C-terminus and/or N-terminus thereof, by1, 2, 3, 4, 5 or more residues, without substantially affecting the pMHCbinding characteristics of the TCR, all such trivial variants areencompassed by the present invention.

As used herein the term “variable region” is understood to encompass allamino acids of a given TCR which are not included within the constantdomain as encoded by the TRAC gene for TCR α chains and either the TRBC1or TRBC2 genes for TCR β chains. (T cell receptor Factsbook, (2001)LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8)

As used herein the term “variable domain” is understood to encompass allamino acids of a given TCR which are encoded by a TRAV gene for TCR αchains and a TRBV gene for TCR β chains. (T cell receptor Factsbook,(2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8)

As is known to those skilled in the art, part of the diversity of theTCR repertoire is due to variations which occur in the amino acidencoded by the codon at the boundary between the variable region, asdefined herein, and the constant domain. For example, the codon that ispresent at this boundary in the parental HIV Gag TCR sequence results inthe presence of the Histidine (H) residue at the C-terminal of thevariable region sequences herein. This Histidine replaces the N-terminalAsparagine (N) residue encoded by the TRAC gene shown in FIG. 8 a.

Embodiments of the invention include mutated TCRs which comprisemutation of one or more of alpha chain variable region amino acidscorresponding to: 95T, 96N, 97S, 98G, and 100A, for example the aminoacids:

95S or G

96A

97H

98D

100S

The numbering used above is the same as that shown in FIG. 1 a and SEQID No: 1

Embodiments of the invention also include TCRs which comprise mutationof one or more of the TCR beta chain variable region amino acidscorresponding to those listed below, are relative to the amino acidoccurring at these positions in the sequence provided for the native HIVGag TCR alpha chain variable region of the native HIV Gag TCR beta chainin FIG. 1 b and SEQ ID No: 2. The amino acids referred to which may bemutated are: 51Y, 52E, 53E and 54E, for example:

51V or A

52R or L

53G

54V

The numbering used above is the same as that shown in FIG. 1 b and SEQID No: 2

Further preferred embodiments of the invention are provided by TCRscomprising one of the mutated alpha chain variable region amino acidsequences shown in FIG. 6 (SEQ ID Nos: 11 to 13). Phenotypically silentvariants of such TCRs also form part of this invention.

Additional preferred embodiments of the invention are provided by TCRscomprising one of the mutated beta chain variable region amino acidsequences shown in FIG. 7. (SEQ ID Nos: 14 and 15). Phenotypicallysilent variants of such TCRs also form part of this invention.

Native TCRs exist in heterodimeric αβ or γδ forms. However, recombinantTCRs consisting of αα or ββ homodimers have previously been shown tobind to peptide MHC molecules. Therefore, one embodiment of theinvention is provided by TCR αα or TCR ββ homodimers.

Further preferred embodiments are provided by TCRs of the inventioncomprising the alpha chain variable region amino acid sequence and thebeta chain variable region amino acid sequence combinations listedbelow, phenotypically silent variants of such TCRs also form part ofthis invention:

Alpha chain variable Beta chain variable region sequence, regionsequence, SEQ ID NO: SEQ ID NO: 1 2 1 14 1 15 11 2 12 2 13 2 12 15 13 1512 14 13 14

In another preferred embodiment TCRs of the invention comprising thevariable regioi combinations detailed above further comprise the alphachain constant domain amino acid sequence shown in FIG. 8 a (SEQ ID NO:19) and one of the beta chain amino acid constant domain sequences shownin FIGS. 8 b and 8 c (SEQ ID NOs: 20 and 21) or phenotypically silentvariants thereof.

As used herein the term “phenotypically silent variants” is understoodto refer to those TCRs which have a K_(D) for the saidSLYNTVATL-HLA-A*0201 complex of less than or equal to 1 μM and/or havean off-rate (k_(off)) of 1×10⁻³ S⁻¹ or slower. For example, as is knownto those skilled in the art, it may be possible to produce TCRs thatincorporate minor changes in the constant domain and/or variable regionsthereof compared to those detailed above without altering the affinityand/or off-rate for the interaction with the SLYNTVATL-HLA-A*0201complex. Such trivial variants are included in the scope of thisinvention. Those TCRs in which one or more conservative substitutionshave been made also form part of this invention.

In one broad aspect, the TCRs of the invention are in the form of eithersingle chain TCRs (scTCRs) or dimeric TCRs (dTCRs) as described in WO04/033685 and WO 03/020763.

A suitable scTCR form comprises a first segment constituted by an aminoacid sequence corresponding to a TCR α chain variable region, a secondsegment constituted by an amino acid sequence corresponding to a TCR βchain variable region sequence fused to the N terminus of an amino acidsequence corresponding to a TCR β chain constant domain extracellularsequence, and a linker sequence linking the C terminus of the firstsegment to the N terminus of the second segment.

Alternatively the first segment may be constituted by an amino acidsequence corresponding to a TCR β chain variable region, the secondsegment may be constituted by an amino acid sequence corresponding to aTCR α chain variable region sequence fused to the N terminus of an aminoacid sequence corresponding to a TCR α chain constant domainextracellular sequence

The above scTCRs may further comprise a disulfide bond between the firstand second chains, said disulfide bond being one which has no equivalentin native αβT cell receptors, and wherein the length of the linkersequence and the position of the disulfide bond being such that thevariable domain sequences of the first and second segments are mutuallyorientated substantially as in native αβT cell receptors.

More specifically the first segment may be constituted by an amino acidsequence corresponding to a TCR α chain variable region sequence fusedto the N terminus of an amino acid sequence corresponding to a TCR αchain constant domain extracellular sequence, the second segment may beconstituted by an amino acid sequence corresponding to a TCR β chainvariable region fused to the N terminus of an amino acid sequencecorresponding to TCR β chain constant domain extracellular sequence, anda disulfide bond may be provided between the first and second chains,said disulfide bond being one which has no equivalent in native αβT cellreceptors.

In the above scTCR forms, the linker sequence may link the C terminus ofthe first segment to the N terminus of the second segment, and may havethe formula —PGGG-(SGGGG)_(n)-P— wherein n is 5 or 6 and P is proline, Gis glycine and S is serine.

(SEQ ID NO: 17) -PGGG-SGGGGSGGGGSGGGGSGGGGSGGGG-P (SEQ ID NO: 18)-PGGG-SGGGGSGGGGSGGGGSGGGGSGGGGSGGGG-P

A suitable dTCR form of the TCRs of the present invention comprises afirst polypeptide wherein a sequence corresponding to a TCR α chainvariable region sequence is fused to the N terminus of a sequencecorresponding to a TCR α chain constant domain extracellular sequence,and a second polypeptide wherein a sequence corresponding to a TCR βchain variable region sequence fused to the N terminus a sequencecorresponding to a TCR β chain constant domain extracellular sequence,the first and second polypeptides being linked by a disulfide bond whichhas no equivalent in native αβT cell receptors.

The first polypeptide may comprise a TCR α chain variable regionsequence is fused to the N terminus of a sequence corresponding to a TCRα chain constant domain extracellular sequence, and a second polypeptidewherein a sequence corresponding to a TCR β chain variable regionsequence is fused to the N terminus a sequence corresponding to a TCR βchain constant domain extracellular sequence, the first and secondpolypeptides being linked by a disulfide bond between cysteine residuessubstituted for Thr 48 of exon 1 of TRAC*01 and Ser 57 of exon 1 ofTRBC1*01 or TRBC2*01 or the non-human equivalent thereof (“TRAC” etc.nomenclature herein as per T cell receptor Factsbook, (2001) LeFranc andLeFranc, Academic Press, ISBN 0-12-441352-8).

The dTCR or scTCR form of the TCRs of the invention may have amino acidsequences corresponding to human αβ TCR extracellular constant domainand variable region sequences, and a disulfide bond may link amino acidresidues of the said constant domain sequences, which disulfide bond hasno equivalent in native TCRs. The disulfide bond is between cysteineresidues corresponding to amino acid residues whose β carbon atoms areless than 0.6 nm apart in native TCRs, for example between cysteineresidues substituted for Thr 48 of exon 1 of TRAC*01 and Ser 57 of exon1 of TRBC1*01 or TRBC2*01 or the non-human equivalent thereof. Othersites where cysteines can be introduced to form the disulfide bond arethe following residues in exon 1 of TRAC*01 for the TCR α chain andTRBC1*01 or TRBC2*01 for the TCR β chain:

TCR α TCR β Native β carbon chain chain separation (nm) Thr 45 Ser 770.533 Tyr 10 Ser 17 0.359 Thr 45 Asp 59 0.560 Ser 15 Glu 15 0.59

In addition to the non-native disulfide bond referred to above, the dTCRor scTCR form of the TCRs of the invention may include a disulfide bondbetween residues corresponding to those linked by a disulfide bond innative TCRs.

The dTCR or scTCR form of the TCRs of the invention preferably does notcontain a sequence corresponding to transmembrane or cytoplasmicsequences of native TCRs.

TCRs of the invention bind strongly to the SLYNTVATL-HLA-A2*0201. TheseTCRs also bind to an altered, but still useful, extent to naturallyoccurring variants of the HIV Gag-derived SLYNTVATL when loaded byHLA-A*0201. Variants of the SLYNTVATL which have been isolated from AIDspatients include the following (Sewell et al., (1997) Eur J Immunol. 27:2323-2329):

SLFNTVATL

SLFNTVAVL

SLSNTVATL

SSFNTVATL

SLLNTVATL

SLYNTIATL

SLYNTIAVL

SLFNTIATL

SLFNTIAVL

SLFNFVATL

The mutated amino acids are underlined.

PEGylated TCR Monomers

In one particular embodiment a TCR of the invention is associated withat least one polyalkylene glycol chain(s). This association may be causein a number of ways known to those skilled in the art. In a preferredembodiment the polyalkylene chain(s) is/are covalently linked to theTCR. In a further embodiment the polyethylene glycol chains of thepresent aspect of the invention comprise at least two polyethylenerepeating units.

Multivalent TCR Complexes

One aspect of the invention provides a multivalent TCR complexcomprising at least two TCRs of the invention. In one embodiment of thisaspect, at least two TCR molecules are linked via linker moieties toform multivalent complexes. Preferably the complexes are water soluble,so the linker moiety should be selected accordingly. Furthermore, it ispreferable that the linker moiety should be capable of attachment todefined positions on the TCR molecules, so that the structural diversityof the complexes formed is minimised. One embodiment of the presentaspect is provided by a TCR complex of the invention wherein the polymerchain or peptidic linker sequence extends between amino acid residues ofeach TCR which are not located in a variable region sequence of the TCR.

Since the complexes of the invention may be for use in medicine, thelinker moieties should be chosen with due regard to their pharmaceuticalsuitability, for example their immunogenicity.

Examples of linker moieties which fulfil the above desirable criteriaare known in the art, for example the art of linking antibody fragments.

There are two classes of linker that are preferred for use in theproduction of multivalent TCR molecules of the present invention. A TCRcomplex of the invention in which the TCRs are linked by a polyalkyleneglycol chain provides one embodiment of the present aspect.

The first are hydrophilic polymers such as polyalkylene glycols. Themost commonly used of this class are based on polyethylene glycol orPEG, the structure of which is shown below.HOCH₂CH₂O(CH₂CH₂O)_(n)—CH₂CH₂OH

Wherein n is greater than two. However, others are based on othersuitable, optionally substituted, polyalkylene glycols includepolypropylene glycol, and copolymers of ethylene glycol and propyleneglycol.

Such polymers may be used to treat or conjugate therapeutic agents,particularly polypeptide or protein therapeutics, to achieve beneficialchanges to the PK profile of the therapeutic, for example reduced renalclearance, improved plasma half-life, reduced immunogenicity, andimproved solubility. Such improvements in the PK profile of thePEG-therapeutic conjugate are believe to result from the PEG molecule ormolecules forming a ‘shell’ around the therapeutic which stericallyhinders the reaction with the immune system and reduces proteolyticdegradation. (Casey et al, (2000) Tumor Targetting 4 235-244) The sizeof the hydrophilic polymer used my in particular be selected on thebasis of the intended therapeutic use of the TCR complex. There arenumerous review papers and books that detail the use of PEG and similarmolecules in pharmaceutical formulations. For example, see Harris (1992)Polyethylene Glycol Chemistry—Biotechnical and Biomedical Applications,Plenum, New York, N.Y. or Harris & Zalipsky (1997) Chemistry andBiological Applications of Polyethylene Glycol ACS Books, Washington,D.C.

The polymer used can have a linear or branched conformation. BranchedPEG molecules, or derivatives thereof, can be induced by the addition ofbranching moieties including glycerol and glycerol oligomers,pentaerythritol, sorbitol and lysine.

Usually, the polymer will have a chemically reactive group or groups inits structure, for example at one or both termini, and/or on branchesfrom the backbone, to enable the polymer to link to target sites in theTCR. This chemically reactive group or groups may be attached directlyto the hydrophilic polymer, or there may be a spacer group/moietybetween the hydrophilic polymer and the reactive chemistry as shownbelow:

Reactive chemistry-Hydrophilic polymer-Reactive chemistry

Reactive chemistry-Spacer-Hydrophilic polymer-Spacer-Reactive chemistry

The spacer used in the formation of constructs of the type outlinedabove may be any organic moiety that is a non-reactive, chemicallystable, chain, Such spacers include, by are not limited to thefollowing:—(CH₂)_(n)— wherein n=2 to 5—(CH₂)₃NHCO(CH₂)₂

A TCR complex of the invention in which a divalent alkylene spacerradical is located between the polyalkylene glycol chain and its pointof attachment to a TCR of the complex provides a further embodiment ofthe present aspect.

A TCR complex of the invention in which the polyalkylene glycol chaincomprises at least two polyethylene glycol repeating units provides afurther embodiment of the present aspect.

There are a number of commercial suppliers of hydrophilic polymerslinked, directly or via a spacer, to reactive chemistries that may be ofuse in the present invention. These suppliers include NektarTherapeutics (CA, USA), NOF Corporation (Japan), Sunbio (South Korea)and Enzon Pharmaceuticals (NJ, USA).

Commercially available hydrophilic polymers linked, directly or via aspacer, to reactive chemistries that may be of use in the presentinvention include, but are not limited to, the following:

PEG linker Source Catalogue Description of PEG Number TCR Monomerattachment 5K linear (Maleimide) Nektar 2D2MOHO1 20K linear (Maleimide)Nektar 2D2MOPO1 20K linear (Maleimide) NOF SUNBRIGHT CorporationME-200MA 20K branched (Maleimide) NOF SUNBRIGHT Corporation GL2-200MA30K linear (Maleimide) NOF SUNBRIGHT Corporation ME-300MA 40K branchedPEG (Maleimide) Nektar 2D3XOTO1 5K-NP linear NOF SUNBRIGHT (for Lysattachment) Corporation MENP-50H 10K-NP linear NOF SUNBRIGHT (for Lysattachment) Corporation MENP-10T 20K-NP linear NOF SUNBRIGHT (for Lysattachment) Corporation MENP-20T TCR dimer linkers 3.4K linear(Maleimide) Nektar 2D2DOFO2 5K forked (Maleimide) Nektar 2D2DOHOF 10Klinear (with orthopyridyl ds- Sunbio linkers in place of Maleimide) 20Kforked (Maleimide) Nektar 2D2DOPOF 20K linear (Maleimide) NOFCorporation 40K forked (Maleimide) Nektar 2D3XOTOF Higher order TCRmultimers 15K, 3 arms, Mal₃ (for trimer) Nektar OJOONO3 20K, 4 arms,Mal₄ (for tetramer) Nektar OJOOPO4 40K, 8 arms, Mal₈ (for octamer)Nektar OJOOTO8

A wide variety of coupling chemistries can be used to couple polymermolecules to protein and peptide therapeutics. The choice of the mostappropriate coupling chemistry is largely dependant on the desiredcoupling site. For example, the following coupling chemistries have beenused attached to one or more of the termini of PEG molecules (Source:Nektar Molecular Engineering Catalogue 2003):

N-maleimide

Vinyl sulfone

Benzotriazole carbonate

Succinimidyl proprionate

Succinimidyl butanoate

Thio-ester

Acetaldehydes

Acrylates

Biotin

Primary amines

As stated above non-PEG based polymers also provide suitable linkers formultimerising the TCRs of the present invention. For example, moietiescontaining maleimide termini linked by aliphatic chains such as BMH andBMOE (Pierce, products Nos. 22330 and 22323) can be used.

Peptidic linkers are the other class of TCR linkers. These linkers arecomprised of chains of amino acids, and function to produce simplelinkers or multimerisation domains onto which TCR molecules can beattached. The biotin/streptavidin system has previously been used toproduce TCR tetramers (see WO/99/60119) for in-vitro binding studies.However, strepavidin is a microbially-derived polypeptide and as suchnot ideally suited to use in a therapeutic.

A TCR complex of the invention in which the TCRs are linked by apeptidic linker derived from a human multimerisation domain provides afurther embodiment of the present aspect.

There are a number of human proteins that contain a multimerisationdomain that could be used in the production of multivalent TCRcomplexes. For example the tetramerisation domain of p53 which has beenutilised to produce tetramers of scFv antibody fragments which exhibitedincreased serum persistence and significantly reduced off-rate comparedto the monomeric scFV fragment. (Willuda et al. (2001) J. Biol. Chem.276 (17) 14385-14392) Haemoglobin also has a tetramerisation domain thatcould potentially be used for this kind of application.

A multivalent TCR complex of the invention comprising at least two TCRsprovides a final embodiment of this aspect, wherein at least one of saidTCRs is associated with a therapeutic agent.

In one aspect a TCR (or multivalent complex thereof) of the presentinvention may alternatively or additionally comprise a reactive cysteineat the C-terminal or N-terminal of the alpha or beta chains thereof.

Diagnostic and Therapeutic Use

In one aspect the TCR of the invention may be associated with atherapeutic agent or detectable moiety. For example, said therapeuticagent or detectable moiety may be covalently linked to the TCR.

In one embodiment of the invention said therapeutic agent or detectablemoiety is covalently linked to the C-terminus of one or both TCR chains.

In one aspect the scTCR or one or both of the dTCR chains of TCRs of thepresent invention may be labelled with an detectable moiety, for examplea label that is suitable for diagnostic purposes. Such labelled TCRs areuseful in a method for detecting a SLYNTVATL-HLA-A*0201 complex whichmethod comprises contacting the TCR ligand with a TCR (or a multimerichigh affinity TCR complex) which is specific for the TCR ligand; anddetecting binding to the TCR ligand. In tetrameric TCR complexes formedfor example, using biotinylated heterodimers, fluorescent streptavidincan be used to provide a detectable label. Such a fluorescently-labelledTCR tetramer is suitable for use in FACS analysis, for example to detectantigen presenting cells carrying the SLYNTVATL-HLA-A*0201 complex forwhich these high affinity TCRs are specific.

Another manner in which the soluble TCRs of the present invention may bedetected is by the use of TCR-specific antibodies, in particularmonoclonal antibodies. There are many commercially available anti-TCRantibodies, such as αFl and βFl, which recognise the constant domains ofthe α and β chains, respectively.

In a further aspect a TCR (or multivalent complex thereof) of thepresent invention may alternatively or additionally be associated with(e.g. covalently or otherwise linked to) a therapeutic agent which maybe, for example, a toxic moiety for use in cell killing, or an immuneeffector molecule such as an interleukin or a cytokine A multivalent TCRcomplex of the invention may have enhanced binding capability for a TCRligand compared to a non-multimeric wild-type or T cell receptorheterodimer of the invention. Thus, the multivalent TCR complexesaccording to the invention are particularly useful for tracking ortargeting cells presenting SLYNTVATL-HLA-A*0201 complexes in vitro or invivo, and are also useful as intermediates for the production of furthermultivalent TCR complexes having such uses. These TCRs or multivalentTCR complexes may therefore be provided in a pharmaceutically acceptableformulation for use in vivo.

The invention also provides a method for delivering a therapeutic agentto a target cell, which method comprises contacting potential targetcells with a TCR or multivalent TCR complex in accordance with theinvention under conditions to allow attachment of the TCR or multivalentTCR complex to the target cell, said TCR or multivalent TCR complexbeing specific for the SLYNTVATL-HLA-A*0201 complex and having thetherapeutic agent associated therewith.

In particular, the soluble TCR or multivalent TCR complex of the presentinvention can be used to deliver therapeutic agents to the location ofcells presenting a particular antigen. This would be useful in manysituations and, in particular, against HIV infected cells. A therapeuticagent could be delivered such that it would exercise its effect locallybut not only on the cell it binds to. Thus, one particular strategyenvisages cytotoxic or immuno-stimulatory molecules linked to TCRs ormultivalent TCR complexes according to the invention specific for theSLYNTVATL-HLA-A*0201 complex.

Many therapeutic agents could be employed for this use, for instanceradioactive compounds, enzymes (perforin for example) orchemotherapeutic agents (cis-platin for example). To ensure that toxiceffects are exercised in the desired location the toxin could be insidea liposome linked to streptavidin so that the compound is releasedslowly. This will prevent damaging effects during the transport in thebody and ensure that the toxin has maximum effect after binding of theTCR to the relevant antigen presenting cells.

Other suitable therapeutic agents include:

-   -   small molecule cytotoxic agents, i.e. compounds with the ability        to kill mammalian cells having a molecular weight of less than        700 daltons. Such compounds could also contain toxic metals        capable of having a cytotoxic effect. Furthermore, it is to be        understood that these small molecule cytotoxic agents also        include pro-drugs, i.e. compounds that decay or are converted        under physiological conditions to release cytotoxic agents.        Examples of such agents include cis-platin, maytansine        derivatives, rachelmycin, calicheamicin, docetaxel, etoposide,        gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone,        sorfimer sodiumphotofrin II, temozolmide, topotecan, trimeterate        glucuronate, auristatin E vincristine and doxorubicin;    -   peptide cytotoxins, i.e. proteins or fragments thereof with the        ability to kill mammalian cells. Including but not limited to,        ricin, diphtheria toxin, pseudomonas bacterial exotoxin A,        DNAase and RNAase;    -   radio-nuclides, i.e. unstable isotopes of elements which decay        with the concurrent emission of one or more of α or β particles,        or γ rays. including but not limited to, iodine 131, rhenium        186, indium 111, yttrium 90, bismuth 210 and 213, actinium 225        and astatine 213; chelating agents may be used to facilitate the        association of these radio-nuclides to the high affinity TCRs,        or multimers thereof;    -   prodrugs, including but not limited to, antibody directed enzyme        pro-drugs;    -   immuno-stimulants, i.e. moieties which stimulate immune        response. Including but not limited to, cytokines such as IL-2        and IFN, Superantigens and mutants thereof, TCR-HLA fusions and        chemokines such as IL-8, platelet factor 4, melanoma growth        stimulatory protein, etc, antibodies or fragments thereof,        complement activators, xenogeneic protein domains, allogeneic        protein domains, viral/bacterial protein domains,        viral/bacterial peptides and anti-T cell determinant antibodies        (e.g. anti-CD3 or anti-CD28) or antibody analogues such as        Nanobodies™ and Affybodies™

Soluble TCRs or multivalent TCR complexes of the invention may be linkedto an enzyme capable of converting a prodrug to a drug. This allows theprodrug to be converted to the drug only at the site where it isrequired (i.e. targeted by the sTCR).

It is expected that the high affinity SLYNTVATL (SEQ ID NO:16)-HLA-A*0201 specific TCRs disclosed herein may be used in methods forthe diagnosis and treatment of AIDS.

For treatment, therapeutic agent localisation in the vicinity of HIVinfected (CD4⁺) cells would enhance the effect of toxins orimmunostimulants. For vaccine delivery, the vaccine antigen could belocalised in the vicinity of antigen presenting cells, thus enhancingthe efficacy of the antigen. The method can also be applied for imagingpurposes.

One embodiment is provided by a membrane preparation comprising a TCR ofthe invention. Said membrane preparation may be prepared from cells ormay comprise a synthetic membrane.

Another embodiment is provided by a cell harbouring an expression vectorcomprising nucleic acid encoding a TCR of the invention. For example,said cell may be a T cell.

Further embodiments of the invention are provided by a pharmaceuticalcomposition comprising:

-   -   a TCR or a multivalent TCR complex of the invention (optionally        associated with a therapeutic agent), or a membrane preparation        comprising a TCR of the invention, or a plurality of cells        harbouring an expression vector comprising nucleic acid encoding        a TCR of the invention, together with a pharmaceutically        acceptable carrier;

The invention also provides a method of treatment of AIDS comprisingadministering to a subject suffering such AIDS an effective amount of aTCR or a multivalent TCR complex of the invention, or a membranepreparation comprising a TCR of the invention, or a plurality of cellsharbouring an expression vector comprising nucleic acid encoding a TCRof the invention. In a related embodiment the invention provides for theuse of a TCR or a multivalent TCR complex of the invention, or amembrane preparation comprising a TCR of the invention, or a pluralityof cells harbouring an expression vector comprising nucleic acidencoding a TCR of the invention, in the preparation of a composition forthe treatment of AIDS. Further specific embodiments of these uses andmethods of the invention are provided wherein the TCR, or multivalentTCR complex of the invention, or a membrane preparation comprising a TCRof the invention is administered in a form which is associated with atherapeutic agent. In other preferred embodiments the cells harbouringan expression vector comprising nucleic acid encoding a TCR of theinvention are CD8⁺ T cells.

Therapeutic or imaging TCRs in accordance with the invention willusually be supplied as part of a sterile, pharmaceutical compositionwhich will normally include a pharmaceutically acceptable carrier. Thispharmaceutical composition may be in any suitable form, (depending uponthe desired method of administering it to a patient). It may be providedin unit dosage form, will generally be provided in a sealed containerand may be provided as part of a kit. Such a kit would normally(although not necessarily) include instructions for use. It may includea plurality of said unit dosage forms.

Without wishing to be limited by theory, it is expected that the TCRs ofthe invention will provide effective targeting agents capable ofdelivering therapeutic agents such as immunostimulants and/or cytotoxicagents to HIV infected (CD4⁺) cells. In particular, it is expected thatthe administration of the TCRs of the present invention when associatedwith immunostimulants and/or cytotoxic agents in combination withconventional anti-retrovirus drug therapies and/or IL-2 treatment willbe able to target HIV infected cells.

The following is a list of anti-retroviral drugs currently approved foruse in the US:

-   -   Agenerase (amprenavir)—protease inhibitor    -   Combivir—combination of Retrovir (300 mg) and Epivir (150 mg)    -   Crixivan (indinavir)—protease inhibitor    -   Epivir (3tc/lamivudine)—nucleoside analog reverse transcriptase        inhibitor    -   Epzicom (a combination of 2 nucleoside reverse transcriptase        inhibitors (NRTIs in the same pill; 600 mg of Ziagen (abacavir)        and 300 mg of Epivir (3TC).    -   Emtriva [emtricitabine (FTC)]    -   Fortovase (saquinavir)—protease inhibitor    -   Fuzeon (enfuvirtide)—Fusion inhibitor    -   Hivid (ddc/zalcitabine)—nucleoside analog reverse transcriptase        inhibitor    -   Invirase (saquinavir)—protease inhibitor    -   Kaletra (lopinavir)—protease inhibitor    -   Lexiva (Fosamprenavir)—Protease Inhibitor approved Oct. 20, 2003    -   Norvir (ritonavir)—protease inhibitor    -   Rescriptor (delavirdine)—non nucleoside analog reverse        transcriptase inhibitor    -   Retrovir, AZT (zidovudine)—nucleoside analog reverse        transcriptase inhibitor    -   Reyataz (atazanavir; BMS-232632)—protease inhibitor    -   Sustiva (efavirenz)—non nucleoside analog reverse transcriptase        inhibitor    -   Trizivir (3 non nucleosides in one tablet;        abacavir+zidovudine+lamivudine    -   Truvada (Emtricitabine+Tenofovir DF)    -   Videx (ddl/didanosine) nucleoside analog reverse transcriptase        inhibitor    -   Videx EC; (ddl/didanosine) nucleoside analog reverse        transcriptase inhibitor;    -   Viracept (nelfinavir)—protease inhibitor    -   Viramune (nevirapine)—non nucleoside analog Reverse        transcriptase inhibitor    -   Viread (tenofovir disoproxil fumarate) Nucleotide Reverse        transcriptase inhibitor (Adenosine Class)    -   Zerit (d4t/stavudine)—nucleoside analog reverse transcriptase        inhibitor    -   Ziagen (abacavir)—nucleoside analog reverse transcriptase        inhibitor

The pharmaceutical composition may be adapted for administration by anyappropriate route, for example parenteral, transdermal or viainhalation, preferably a parenteral (including subcutaneous,intramuscular, or, most preferably intravenous) route. Such compositionsmay be prepared by any method known in the art of pharmacy, for exampleby mixing the active ingredient with the carrier(s) or excipient(s)under sterile conditions.

Dosages of the substances of the present invention can vary between widelimits, depending upon the disease or disorder to be treated, the ageand condition of the individual to be treated, etc. and a physician willultimately determine appropriate dosages to be used.

Additional Aspects

A scTCR or dTCR (which preferably is constituted by constant andvariable sequences corresponding to human sequences) of the presentinvention may be provided in substantially pure form, or as a purifiedor isolated preparation. For example, it may be provided in a form whichis substantially free of other proteins.

The sequence(s) of the nucleic acid or nucleic acids encoding the TCRsof the invention may be altered so as to optimise the level ofexpression obtained in the host cell. The host cell may be anyappropriate prokaryotic or eukaryotic cell. For example, the host cellmay be an E. coli cell or a human T cell. The alterations made to thesegenetic sequences are silent, that is they do not alter the amino acidsequence encoded. There are a number of companies which offer suchexpression optimisation services, including, GeneArt, Germany.

The invention also provides a method of producing a high affinity TCRhaving the property of binding to SLYNTVATL-HLA-A*0201.CHARACTERISED INTHAT the TCR (i) comprises at least one TCR α chain variable domainand/or at least one TCR β chain variable domain and (ii) has a K_(D) forthe said SLYNTVATL-HLA-A*0201 complex of less than or equal to 1 μMand/or an off-rate (k_(off)) for the SLYNTVATL-HLA-A*0201 complex of1×10⁻³ S⁻¹ or slower, wherein the method comprises:

-   -   (a) the production of a TCR comprising the α and β chain        variable domains of the parental HIV Gag TCR wherein one or both        of the α and β chain variable domains comprise a mutation(s) in        one or more of the amino acids identified in claims 7 and 8;    -   (b) contacting said mutated TCR with SLYNTVATL-HLA-A*0201 under        conditions suitable to allow the binding of the TCR to        SLYNTVATL-HLA-A*0201;        and measuring the K_(D) and/or k_(off) of the interaction.

Preferred features of each aspect of the invention are as for each ofthe other aspects mutatis mutandis. The prior art documents mentionedherein are incorporated to the fullest extent permitted by law.

The invention is further described in the following examples, which donot limit the scope of the invention in any way.

EXAMPLES Example 1 Production of Soluble Disulfide-Linked TCRsComprising the Parental HIV Gag TCR Variable Regions

FIGS. 4 a and 4 b provide the DNA sequences of soluble disulfide-linkedalpha beta chains from a parental TCR which is specific for theSLYNTVATL-HLA-A*0201 complex. These DNA sequences can be synthesisde-novo by a number of contract research companies, for example GeneArt(Germany). Restriction enzyme recognition sites are also added to theseDNA sequences in order to facilitate ligation of these DNA sequencesinto the pGMT7-based expression plasmids, which contain the T7 promoterfor high level expression in E. coli strain BL21-DE3(pLysS) (Pan et al.,Biotechniques (2000) 29 (6): 1234-8)

The TCR alpha chain sequences contain introduced ClaI and SalIrestriction enzyme recognition sites and this sequence was ligated intopEX954 (see FIGS. 9 and 13) cut with ClaI and XhoI.

The TCR beta chain sequences contain introduced AseI and AgeIrestriction enzyme recognition sites and were ligated into pEX821 (seeFIGS. 10 and 14) cut with NdeI/AgeI.

Restriction enzyme recognition sites as introduced into DNA encoding theTCR chains

ClaI—ATCGAT

SalI—GTCGAC

AseI—ATTAAT

AgeI—ACCGGT

Ligation

The cut TCR alpha and beta chain DNA and cut vector were ligated using arapid DNA ligation kit (Roche) following the manufacturers instructions.

Ligated plasmids were transformed into competent E. coli strain XL1-bluecells and plated out on LB/agar plates containing 100 mg/ml ampicillin.Following incubation overnight at 37° C., single colonies were pickedand grown in 10 ml LB containing 100 mg/ml ampicillin overnight at 37°C. with shaking Cloned plasmids were purified using a Miniprep kit(Qiagen) and the insert was sequenced using an automated DNA sequencer(Lark Technologies).

FIGS. 5 a and 5 b show respectively the soluble disulfide linkedparental HIV gag TCR α and β chain extracellular amino acid sequencesproduced from the DNA sequences of FIGS. 4 a and 4 b

Example 2 Production of High Affinity Variants of the Soluble DisulfideLinked HIV Gag TCR

The soluble disulfide-linked native HIV Gag TCR produced as described inExample 1 can be used a template from which to produce the TCRs of theinvention which have an increased affinity for the SLYNTVATL (SEQ ID NO:16)-HLA-A*0201 complex.

Phage display is one means by which libraries of HIV Gag TCR variantscan be generated in order to identify high affinity mutants. Forexample, the TCR phage display and screening methods described in (Li etal., (2005) Nature Biotech 23 (3): 349-354) can be adapted and appliedto HIV Gag TCRs.

The amino sequences of the mutated TCR alpha and beta chain variabledomains which, when combined with an appropriate TCR chain, demonstratehigh affinity for the SLYNTVATL-HLA-A*0201 complex, are listed in FIGS.6 and 7 respectively. (SEQ ID Nos: 11-13 and 14-15 respectively) As isknown to those skilled in the art the necessary codon changes requiredto produce these mutated chains can be introduced into the DNA encodingthese chains by site-directed mutagenesis. (QuickChange™ Site-DirectedMutagenesis Kit from Stratagene)

Briefly, this is achieved by using primers that incorporate the desiredcodon change(s) and the plasmids containing the relevant TCR chain DNAas a template for the mutagenesis:

Mutagenesis was carried out using the following conditions: 50 ngplasmid template, 1 μl of 10 mM dNTP, 5 μl of 10×Pfu DNA polymerasebuffer as supplied by the manufacturer, 25 pmol of fwd primer, 25 pmolof rev primer, 1 μl pfu DNA polymerase in total volume 50 μl. After aninitial denaturation step of 2 mins at 95 C, the reaction was subjectedto 25 cycles of denaturation (95 C, 10 secs), annealing (55 C 10 secs),and elongation (72 C, 8 mins). The resulting product was digested withDpnI restriction enzyme to remove the template plasmid and transformedinto E. coli strain XL1-blue. Mutagenesis was verified by sequencing.

Example 3 Expression, Refolding and Purification of Soluble TCR

The expression plasmids containing the mutated α-chain and β-chainrespectively as prepared in Examples 1 or 2 were transformed separatelyinto E. coli strain BL2pLysS, and single ampicillin-resistant colonieswere grown at 37° C. in TYP (ampicillin 100 μg/ml) medium to OD₆₀₀ of0.4 before inducing protein expression with 0.5 mM IPTG. Cells wereharvested three hours post-induction by centrifugation for 30 minutes at4000 rpm in a Beckman J-6B. Cell pellets were re-suspended in a buffercontaining 50 mM Tris-HCl, 25% (w/v) sucrose, 1 mM NaEDTA, 0.1% (w/v)NaAzide, 10 mM DTT, pH 8.0. After an overnight freeze-thaw step,re-suspended cells were sonicated in 1 minute bursts for a total ofaround 10 minutes in a Milsonix XL2020 sonicator using a standard 12 mmdiameter probe. Inclusion body pellets were recovered by centrifugationfor 30 minutes at 13000 rpm in a Beckman J2-21 centrifuge. Threedetergent washes were then carried out to remove cell debris andmembrane components. Each time the inclusion body pellet was homogenisedin a Triton buffer (50 mM Tris-HCl, 0.5% Triton-X100, 200 mM NaCl, 10 mMNaEDTA, 0.1% (w/v) NaAzide, 2 mM DTT, pH 8.0) before being pelleted bycentrifugation for 15 minutes at 13000 rpm in a Beckman J2-21. Detergentand salt was then removed by a similar wash in the following buffer: 50mM Tris-HCl, 1 mM NaEDTA, 0.1% (w/v) NaAzide, 2 mM DTT, pH 8.0. Finally,the inclusion bodies were divided into 30 mg aliquots and frozen at −70°C. Inclusion body protein yield was quantitated by solubilising with 6Mguanidine-HCl and measurement with a Bradford dye-binding assay(PerBio).

Approximately 30 mg of TCR β chain and 60 mg of TCR α chain solubilisedinclusion bodies were thawed from frozen stocks, samples were then mixedand the mixture diluted into 15 ml of a guanidine solution (6 MGuanidine-hydrochloride, 10 mM Sodium Acetate, 10 mM EDTA), to ensurecomplete chain de-naturation. The guanidine solution containing fullyreduced and denatured TCR chains was then injected into 1 liter of thefollowing refolding buffer: 100 mM Tris pH 8.5, 400 mM L-Arginine, 2 mMEDTA, 5 mM reduced Glutathione, 0.5 mM oxidised Glutathione, 5M urea,0.2 mM PMSF. The redox couple (2-mercaptoethylamine and cystamine (tofinal concentrations of 6.6 mM and 3.7 mM, respectively) were addedapproximately 5 minutes before addition of the denatured TCR chains. Thesolution was left for 5 hrs±15 minutes. The refolded TCR was dialysed inSpectrapor 1 membrane (Spectrum; Product No. 132670) against 10 L 10 mMTris pH 8.1 at 5° C.±3° C. for 18-20 hours. After this time, thedialysis buffer was changed to fresh 10 mM Tris pH 8.1 (10 L) anddialysis was continued at 5° C.±3° C. for another 20-22 hours.

sTCR was separated from degradation products and impurities by loadingthe dialysed refold onto a POROS 50HQ anion exchange column and elutingbound protein with a gradient of 0-500 mM NaCl over 50 column volumesusing an Akta purifier (Pharmacia). Peak fractions were stored at 4° C.and analysed by Coomassie-stained SDS-PAGE before being pooled andconcentrated. Finally, the sTCR was purified and characterised using aSuperdex 200HR gel filtration column pre-equilibrated in HBS-EP buffer(10 mM HEPES pH 7.4, 150 mM NaCl, 3.5 mM EDTA, 0.05% nonidet p40). Thepeak eluting at a relative molecular weight of approximately 50 kDa waspooled and concentrated prior to characterisation by BIAcore surfaceplasmon resonance analysis.

Example 4 Biacore Surface Plasmon Resonance Characterisation of sTCRBinding to Specific pMHC

A surface plasmon resonance biosensor (Biacore 3000™) was used toanalyse the binding of a sTCR to its peptide-MHC ligand. This wasfacilitated by producing single pMHC complexes (described below) whichwere immobilised to a streptavidin-coated binding surface in asemi-oriented fashion, allowing efficient testing of the binding of asoluble T-cell receptor to up to four different pMHC (immobilised onseparate flow cells) simultaneously. Manual injection of HLA complexallows the precise level of immobilised class I molecules to bemanipulated easily.

Biotinylated class I HLA-A*0201 molecules were refolded in vitro frombacterially-expressed inclusion bodies containing the constituentsubunit proteins and synthetic peptide, followed by purification and invitro enzymatic biotinylation (O'Callaghan et al. (1999) Anal. Biochem.266: 9-15). HLA-A*0201-heavy chain was expressed with a C-terminalbiotinylation tag which replaces the transmembrane and cytoplasmicdomains of the protein in an appropriate construct. Inclusion bodyexpression levels of ˜75 mg/liter bacterial culture were obtained. TheMHC light-chain or β2-microglobulin was also expressed as inclusionbodies in E. coli from an appropriate construct, at a level of ˜500mg/liter bacterial culture.

E. coli cells were lysed and inclusion bodies are purified toapproximately 80% purity. Protein from inclusion bodies was denatured in6 M guanidine-HCl, 50 mM Tris pH 8.1, 100 mM NaCl, 10 mM DTT, 10 mMEDTA, and was refolded at a concentration of 30 mg/liter heavy chain, 30mg/liter β2m into 0.4 M L-Arginine-HCl, 100 mM Tris pH 8.1, 3.7 mMcystamine, 6.6 mM β-cysteamine, 4 mg/ml of the SLYNTVATL peptiderequired to be loaded by the HLA-A*0201 molecule, by addition of asingle pulse of denatured protein into refold buffer at <5° C. Refoldingwas allowed to reach completion at 4° C. for at least 1 hour.

Buffer was exchanged by dialysis in 10 volumes of 10 mM Tris pH 8.1. Twochanges of buffer were necessary to reduce the ionic strength of thesolution sufficiently. The protein solution was then filtered through a1.5 μm cellulose acetate filter and loaded onto a POROS 50HQ anionexchange column (8 ml bed volume). Protein was eluted with a linear0-500 mM NaCl gradient. HLA-A*0201-peptide complex eluted atapproximately 250 mM NaCl, and peak fractions were collected, a cocktailof protease inhibitors (Calbiochem) was added and the fractions werechilled on ice.

Biotinylation tagged pMHC molecules were buffer exchanged into 10 mMTris pH 8.1, 5 mM NaCl using a Pharmacia fast desalting columnequilibrated in the same buffer. Immediately upon elution, theprotein-containing fractions were chilled on ice and protease inhibitorcocktail (Calbiochem) was added. Biotinylation reagents were then added:1 mM biotin, 5 mM ATP (buffered to pH 8), 7.5 mM MgCl2, and 5 μg/ml BirAenzyme (purified according to O'Callaghan et al. (1999) Anal. Biochem.266: 9-15). The mixture was then allowed to incubate at room temperatureovernight.

The biotinylated pHLA-A*0201 molecules were purified using gelfiltration chromatography. A Pharmacia Superdex 75 HR 10/30 column waspre-equilibrated with filtered PBS and 1 ml of the biotinylationreaction mixture was loaded and the column was developed with PBS at 0.5ml/min. Biotinylated pHLA-A*0201 molecules eluted as a single peak atapproximately 15 ml. Fractions containing protein were pooled, chilledon ice, and protease inhibitor cocktail was added. Protein concentrationwas determined using a Coomassie-binding assay (PerBio) and aliquots ofbiotinylated pHLA-A*0201 molecules were stored frozen at −20° C.Streptavidin was immobilised by standard amine coupling methods.

Such immobilised complexes are capable of binding both T-cell receptorsand the coreceptor CD8αα, both of which may be injected in the solublephase. Specific binding of TCR is obtained even at low concentrations(at least 40 μg/ml), implying the TCR is relatively stable. The pMHCbinding properties of sTCR are observed to be qualitatively andquantitatively similar if sTCR is used either in the soluble orimmobilised phase. This is an important control for partial activity ofsoluble species and also suggests that biotinylated pMHC complexes arebiologically as active as non-biotinylated complexes.

The interactions between HIV Gag sTCR containing a novel inter-chainbond and its ligand/MHC complex or an irrelevant HLA-peptidecombination, the production of which is described above, were analysedon a Biacore 3000™ surface plasmon resonance (SPR) biosensor. SPRmeasures changes in refractive index expressed in response units (RU)near a sensor surface within a small flow cell, a principle that can beused to detect receptor ligand interactions and to analyse theiraffinity and kinetic parameters. The probe flow cells were prepared byimmobilising the individual HLA-peptide complexes in separate flow cellsvia binding between the biotin cross linked onto β2m and streptavidinwhich have been chemically cross linked to the activated surface of theflow cells. The assay was then performed by passing sTCR over thesurfaces of the different flow cells at a constant flow rate, measuringthe SPR response in doing so.

To Measure Equilibrium Binding Constant

Serial dilutions of the parental or mutated HIV Gag sTCR were preparedand injected at constant flow rate of 5 μl min-1 over two different flowcells; one coated with ˜1000 RU of specific SLYNTVATL-HLA-A*0201complex, the second coated with ˜1000 RU of non-specific HLA-A2-peptidecomplex. Response was normalised for each concentration using themeasurement from the control cell. Normalised data response was plottedversus concentration of TCR sample and fitted to a hyperbola in order tocalculate the equilibrium binding constant, K_(D). (Price & Dwek,Principles and Problems in Physical Chemistry for Biochemists (2^(nd)Edition) 1979, Clarendon Press, Oxford).

To Measure Kinetic Parameters

For high affinity TCRs K_(D) was determined by experimentally measuringthe dissociation rate constant, kd, and the association rate constant,ka. The equilibrium constant K_(D) was calculated as kd/ka.

TCR was injected over two different cells one coated with ˜300 RU ofspecific HLA-A2-nyeso peptide complex, the second coated with ˜300 RU ofnon-specific HLA-A2-peptide complex. Flow rate was set at 50 μl/min.Typically 250 μA of TCR at ˜3 μM concentration was injected. Buffer wasthen flowed over until the response had returned to baseline. Kineticparameters were calculated using Biaevaluation software. Thedissociation phase was also fitted to a single exponential decayequation enabling calculation of half-life.

Results

The interaction between a soluble disulfide-linked native HIV Gag TCR(consisting of the α and β TCR chains detailed in SEQ ID NOs 9 and 10respectively) and the SLYNTVATL-HLA-A*0201 complex was analysed usingthe above methods and demonstrated a K_(D) of 85 nM and an off-rate(k_(off)) of 2.21×10⁻² S⁻¹. (See FIG. 12 for Biacore response curves)

The TCRs specified in the following table have a K_(D) of less than orequal to 1 μM and/or a k_(off) of 1×10⁻³ S⁻¹ or slower.

Alpha chain variable Beta chain variable domain sequence, domainsequence, SEQ ID NO: SEQ ID NO: 1 2 1 14 1 15 11 2 12 2 13 2 12 15 13 1512 14 13 14

Example 5 Production of a Soluble High Affinity HIV Gag TCR-WT HumanIL-2 Fusion Protein

The methods substantially as described in Examples 1 to 3 can be used toproduce a soluble high affinity HIV Gag TCR-WT human IL-2 fusionprotein. Briefly, the DNA encoding the desired linker and WT human IL-2are added into the 3′ end of the DNA sequence of the solubledisulfide-linked parental HIV Gag TCR beta chain immediately prior tothe TAA (“Stop”) codon. FIG. 11 provides the amino acid sequence of afusion protein comprising a disulfide-linked parental HIV Gag TCR betachain fused to WT human IL-2 via linker sequence. (SEQ ID NO: 24) Thelinker and IL-2 portion of this fusion protein are indicated in italics.The DNA encoding this construct can then be ligated into pEX821. Thesoluble parental HIV Gag TCR-IL-2 fusion protein can then be expressedby combining this beta chain fusion protein with the solubledisulfide-linked parental HIV Gag alpha chain TCR chain detailed in FIG.5 a (SEQ ID NO: 9) using the methods substantially as described inExample 3.

Example 6 Recombinant Expression of the Parental HIV Gag TCR on theSurface of T Cells

DNA constructs encoding the signal sequence, extracellular,transmembrane and intracellular domains of the parental HIV Gag TCRchains were synthesised (GeneArt, Germany). These TCR α chain and TCR βchain DNA sequences, provided in FIGS. 15 a and 15 b respectively, arealtered from the parental HIV Gag TCR DNA sequences so as to enhanceexpression levels of the encoded TCR chains in human T cells whilstmaintaining the native amino acid sequence. FIGS. 16 a and 16 b providethe full-length amino acid sequences encoded by the DNA sequences ofFIGS. 15 a and 15 b respectively.

TCR α chain and TCR β chain DNA sequences were then inserted togetherinto a Lentiviral expression vector. This vector contains DNA encodingboth the parental HIV Gag TCR α chain and β chain as a single openreading frame with the in-frame Foot and Mouth Disease Virus (FMDV) 2Acleavage factor amino acid sequence (LLNFDLLKLAGDVESNPG (SEQ ID NO: 31))separating the TCR chains. (de Felipe et al., Genet Vaccines Ther (2004)2 (1): 13) On mRNA translation the TCR α chain is produced with the 2Apeptide sequence at its C-terminus and the TCR β chain is produced as aseparate polypeptide.

T cells were transduced with the above Lentiviral vector. Briefly,primary T cells were stimulated for 24 hours using anti-CD3/anti-CD28beads. A concentrated Lentivirus supernatant, expressing the TCR genes,was then incubated with the stimulated T cells to allow viraltransduction. The anti-CD3/anti-CD28 beads were then removed and thetransduced T cells were cultured until they attained a “resting volume”of 200-300 fL.

Presentation of parental HIV Gag TCRs on the surface of the transducedcells was confirmed by FACS analysis using HLA-A*0201-SLYNTVALT PEtetramer and anti-CD8 monoclonal antibody FITC co-staining.

Results

FIG. 17 b provides the FACS analysis data which demonstrates thesuccessful expression of the parental HIV Gag TCR on the surface oftransduced CD8⁺ T cells. FIG. 17 a provides FACS analysis data generatedusing control untransduced T cells.

Example 7 Inhibition of CTL Activation by Soluble High Affinity HIV GagTCRs

The following assays were carried out to demonstrate that the solublehigh affinity c11c6 HIV Gag TCR was capable of inhibiting activation ofa SLYNTVATL-HLA-A*0201 reactive polyclonal T cell line.

Inhibition of Activation of the OX84 SLYNTVATL-HLA-A*0201 ReactivePolyclonal T Cell Line in the of Presence of HIV Infected Cells

The soluble c11c6 high affinity HIV Gag TCR utilised in this experimentcontained the TCR alpha chain variable domain and TCR beta chainvariable regions shown in FIG. 6 c (SEQ ID NO: 13) and FIG. 7 b (SEQ IDNO: 15) respectively. The full amino acid sequences of the TCR alpha andbeta chains of this soluble TCR are provided by FIG. 18 a (SEQ ID NO:29) and FIG. 18 b (SEQ ID NO: 30) respectively.

IFN-γ and TNF-α production was used as the read-outs for CTL activation.

Reagents

R10 Assay media: 10% FCS (heat-inactivated, Gibco, cat#10108-165), 88%RPMI 1640 (Gibco, cat#42401-018), 1% glutamine (Gibco, cat#25030-024)and 1% penicillin/streptomycin (Gibco, cat#15070-063).

Peptide: (obtained from various sources) initially dissolved in DMSO(Sigma, cat# D2650) at 4 mg/ml and frozen.

The BD™ Cytometric Bead Array Kit, Human Th1/Th2 cytokine Kit II (BDBiosciences, San Diego, US) contains all the reagents required for theassay.

T Cell Activation Assay

Chronically HIV infected To target cells (HXB2 and HIV3B HIV Labstrains) were washed and re-suspended in R10 media. As a controluninfected To target cells were pulsed with 1 nM of SLYNTVATL peptide,for 30 minutes at 37° C., 5% CO₂.

Test Samples:

25,000 HIV infected To target cells in R10 media per well of a 96 wellU-bottom plate.

2×10⁻⁷ M high affinity c11c6 HIV Gag TCR or parental HIV Gag TCR in R10media per well.

5000 OX84 polyclonal effector T cell line in R10 media per well.

Controls:

As above substituting irrelevant soluble TCRs (HLA-A*0201-Tax specificand HLA-A*0201-NY-ESO specific TCRs) or the high affinity HIV Gag TCRs.

The plate was then incubated for 4 hours at 37° C., 5% CO₂. The culturesupernatant was removed to measure the levels of IFN-γ and TNF-α presentusing the following method.

IFN-γ and TNF-α Assay

BD™ Cytometric Beads coated with (a) anti-IFNγ capture antibodies and(b) anti-TNFα capture antibodies were prepared according to themanufacturers instructions

A number of assay tubes were then prepared containing the followingadditions:

-   -   50 μl of mixed anti-IFNγ and anti-TNFα BD™ Cytometric Beads in        BD Assay Diluent    -   50 μl of PE Dectection Reagent

Followed by either:

-   -   50 μl of the culture supernatant taken from the T cell        activation assay wells. (Test Samples)    -   Or    -   50 μl of mixed IFNγ and TNFα standards prepared at a range of        concentrations by serial dilution of stock standards.        (Calibration Standards)

The tube were then incubated in the dark for 3 hours prior to beingwashed with 1 ml of BD Wash Buffer and centrifuged. Finally, the beadswere re-suspended in 300 μl of the Wash Buffer and the level of IFNγ andTNFα present was determined by Flow Cytometry according tomanufacturer's instructions.

Inhibition of the SLYNTVATL-HLA-A*0201 Specific OX84 Polyclonal T Linein the Presence of Uninfected SLYNTVATL Peptide Pulsed To Cells

The same regents and methods as used for the above CTL activation assaywere used except that:

2000 OX84 polyclonal effector T cells were used in each T cellactivation assay.

Uninfected To lymphoblastoid cells, pulsed with 10⁻¹⁰−10⁻⁸ M SLYNTVATLpeptide were used as the target cells

Results

The soluble high affinity c11c6 HIV Gag TCR strongly inhibitedactivation of the SLYNTVATL-HLA-A*0201 reactive OX84 polyclonal T cellline in the presence of To cells infected by HIV as measured by IFN-γand TNF-α production. (See FIG. 19)

The soluble high affinity c11c6 HIV Gag TCR strongly inhibitedactivation of the SLYNTVATL-HLA-A*0201 reactive OX84 polyclonal T cellline in the presence of SLYNTVATL-pulsed uninfected To cells as measuredby IFN-γ and TNF-α production. (See FIG. 20)

Example 8 Quantification of Cell Surface SLYNTVATL-HLA-A*0201 Antigenson Peptide Pulsed T2 Cells by Fluorescence Microscopy Using HighAffinity c11c6 HIV Gag TCR

The number of SLYNTVATL-HLA-A*0201 antigens on peptide-pulsed T2lymphoblastoid cell was determined (on the assumption that onefluorescence signal relates to a single labelled TCR bound to itscognate pMHC ligand on the surface of the target cell) by singlemolecule fluorescence microscopy using a soluble high-affinity c11c6 HIVGag TCR. This was facilitated by using biotinylated TCR to target theantigen-expressing cancer cells and subsequent labelling of cell-boundTCR by streptavidin-R phycoerythrin (PE) conjugates. Individual PEmolecules were then imaged by 3-dimensional fluorescence microscopy.

T2 lymphoblastoid cells were pulsed with the HIV Gag-derived SLYNTVATLpeptide, or an irrelevant peptide (SLLMWITQC) at a range ofconcentrations (10⁻⁵−10⁻¹⁰M) for 90 minutes at 37° C. After pulsing thecells were washed twice with 500 μl of PBS. Cells were incubated in 200μl of TCR solution (100 nM high-affinity c11c6 HIV Gag TCR), in PBS.0.5% BSA albumin) for 30 min at room temperature. TCR solution wasremoved, and cells were washed three times with 500 μl of PBS. Cellswere incubated in 200 μl of streptavidin-PE solution (5 μg ml⁻¹streptavidin-PE in PBS containing 0.5% BSA) at room temperature in thedark for 20 min. Streptavidin-PE solution was removed and cells werewashed three times with 500 μl of PBS. Wash media was removed, and cellskept in 400 μl of R10, without Phenol Red before imaging by fluorescencemicroscopy.

Fluorescence Microscopy

Fluorescent microscopy was carried out using an Axiovert 200M (Zeiss)microscope with a 63× Oil objective (Zeiss). A Lambda LS light sourcecontaining a 300 W Xenon Arc lamp (Sutter) was used for illumination,and light intensity was reduced to optimal levels by placing a 0.3 and a0.6 neutral density filter into the light path. Excitation and emissionspectra were separated using a TRITC/DiI filter set (Chroma). Cells wereimaged in three dimensions by z-stack acquisition (21 planes, 1 μmapart). Image acquisition and analysis was performed using Metamorphsoftware (Universal Imaging) as described (Irvine et al., Nature 419:p845-9, and Purbhoo et al., Nature Immunology 5: p524-30).

Results

As shown by FIG. 21 the above method was used successfully to image highaffinity c11c6 HIV Gag TCR bound to SLYNTVATL-HLA-A*0201 antigens on thesurface of peptide-pulsed T2 cells. These results show the threshold forcounting epitopes on SLYNTVATL peptide-pulsed cells using the highaffinity c6c11 HIV Gag TCR is approximately 10⁻⁹ M peptide.

The invention claimed is:
 1. A cell transduced with an expression vectorcomprising nucleic acid encoding a T-cell receptor (TCR) comprising an αchain variable domain and a β chain variable domain wherein: the TCRbinds to SLYNTVATL (SEQ ID NO: 16)-HLA-A*0201 with a K_(D) of less thanor equal to 1 μM, and the α chain variable domain comprises SEQ ID NO:1, and the β chain variable domain comprises SEQ ID NO:
 2. 2. Apharmaceutical composition comprising a plurality of cells as claimed inclaim 1, together with a pharmaceutically acceptable carrier.
 3. A celltransduced with an expression vector comprising nucleic acid encoding aTCR comprising an α chain variable domain and a β chain variable domainwherein: the TCR binds to SLYNTVATL (SEQ ID NO: 16)-HLA-A*0201 with aK_(D) of less than or equal to 1 μM and/or an off-rate (k_(off)) of1×10⁻³ S⁻¹ or slower using Surface Plasmon Resonance, and the α chainvariable domain comprises SEQ ID NO: 1 with at least one mutation in atleast one complementarity determining region selected from the groupconsisting of at least one of 95T, 96N, 97S, 98G and 100A, or the βchain variable domain comprises SEQ ID NO: 2 with at least one mutationin at least one complementarity determining region selected from thegroup consisting of at least one of 51Y, 52E, 53E and 54E wherein: ifthe α chain variable domain is mutated, the β chain variable domaincomprises SEQ ID NO: 2, and if the β chain variable domain is mutated,the α chain variable domain comprises SEQ ID NO:
 1. 4. A cell transducedwith an expression vector comprising nucleic acid encoding a TCRcomprises an α chain variable domain and a β chain variable domainwherein: the TCR binds to SLYNTVATL (SEQ ID NO: 16)-HLA-A*0201 with aK_(D) of less than or equal to 1 μM and/or an off-rate (k_(off)) of1×10⁻³ S⁻¹ or slower using Surface Plasmon Resonance, and the α chainvariable domain comprises SEQ ID NO: 1 with at least one mutation in atleast one complementarity determining region selected from the groupconsisting of, 95T, 96N, 97S, 98G and 100A, and the β chain variabledomain comprises SEQ ID NO:2 with at least one mutation in at least onecomplementarity determining region selected from the group consistingof, 51 Y, 52E, 53E and 54E.
 5. The cell of claim 4 wherein the TCRcomprises the α chain variable domain wherein all of 95T, 96N, 97S, 98Gand 100A are mutated, and the β chain variable domain wherein all of51Y, 52E, 53E or 54E are mutated.
 6. A cell transduced with anexpression vector comprising nucleic acid encoding a TCR comprising an αchain variable domain and a β chain variable domain wherein: the TCRbinds to SLYNTVATL (SEQ ID NO: 16)-HLA-A*0201 with a K_(D) of less thanor equal to 1 μM and/or an off-rate (k_(off)) of 1×10⁻³ S⁻¹ or slowerusing Surface Plasmon Resonance, and the α chain variable domaincomprises SEQ ID NO: 1 with one or more of amino acids 95S, 95G, 96A,97H, 98D or 100S, and is hence mutated relative to SEQ ID NO:1, and theβ chain variable domain comprises SEQ ID NO:2 with one or more of aminoacids 51V, 51A, 52R, 52L, 53G or 54V, and is hence mutated relative toSEQ ID NO:2.
 7. The cell of claim 6 wherein α chain variable domaincomprises amino acids 95S, 95G, 96A, 97H, 98D and 100S, mutated relativeto SEQ ID NO: 1; and the β chain variable domain comprises amino acids51V, 51A, 52R, 52L, 53G and 54V, mutated relative to SEQ ID NO:
 2. 8. Acell transduced with an expression vector comprising nucleic acidencoding a TCR comprising an α chain variable domain and a β chainvariable domain wherein: the TCR binds to SLYNTVATL (SEQ ID NO:16)-HLA-A*0201 with a K_(D) of less than or equal to 1 μM and/or anoff-rate (k_(off)) of 1×10⁻³ S⁻¹ or slower using Surface PlasmonResonance, and the α chain variable domain comprises the amino acidsequence shown in any one of SEQ ID NOS: 11-13, and the β chain variabledomain comprises the amino acid sequence shown in any one of SEQ ID NOS:14-15.
 9. A cell transduced with an expression vector comprising nucleicacid encoding a TCR comprising an α chain variable domain and a β chainvariable domain wherein: the TCR binds to SLYNTVATL (SEQ ID NO:16)-HLA-A*0201 with a K_(D) of less than or equal to 1 μM and/or anoff-rate (k_(off)) of 1×10″³ S⁻¹ or slower using Surface PlasmonResonance, and the α chain variable domain comprises the amino acidsequence shown in SEQ ID NO: 1 and the βchain variable domain comprisesthe amino acid sequence shown in SEQ ID NO: 14; or the α chain variabledomain comprises the amino acid sequence shown in SEQ ID NO: 1 and the βchain variable domain comprises the amino acid sequence shown in SEQ IDNO: 15; or the α chain variable domain comprises the amino acid sequenceshown in SEQ ID NO: 11 and the β chain variable domain comprises theamino acid sequence shown in SEQ ID NO: 2; or the α chain variabledomain comprises the amino acid sequence shown in SEQ ID NO: 12 and theβ chain variable domain comprises the amino acid sequence shown in SEQID NO: 2; or the α chain variable domain comprises the amino acidsequence shown in SEQ ID NO: 13 and the β chain variable domaincomprises the amino acid sequence shown in SEQ ID NO: 2; or the α chainvariable domain comprises the amino acid sequence shown in SEQ ID NO: 12and the β chain variable domain comprises the amino acid sequence shownin SEQ ID NO: 15; or the α chain variable domain comprises the aminoacid sequence shown in SEQ ID NO: 13 and the β chain variable domaincomprises the amino acid sequence shown in SEQ ID NO: 15; or the α chainvariable domain comprises the amino acid sequence shown in SEQ ID NO: 12and the β chain variable domain comprises the amino acid sequence shownin SEQ ID NO: 14; or the α chain variable domain comprises the aminoacid sequence shown in SEQ ID NO: 13 and the β chain variable domaincomprises the amino acid sequence shown in SEQ ID NO:
 14. 10. The cellof claim 9 wherein the α chain variable domain comprises the amino acidsequence shown in SEQ ID NO: 1 and the β chain variable domain comprisesthe amino acid sequence shown in SEQ ID NO:
 14. 11. The cell of claim 9wherein the α chain variable domain comprises the amino acid sequenceshown in SEQ ID NO: 1 and the β chain variable domain comprises theamino acid sequence shown in SEQ ID NO:
 15. 12. The cell of claim 9wherein the α chain variable domain comprises the amino acid sequenceshown in SEQ ID NO: 11 and the β chain variable domain comprises theamino acid sequence shown in SEQ ID NO:
 2. 13. The cell of claim 9wherein the α chain variable domain comprises the amino acid sequenceshown in SEQ ID NO: 12 and the β chain variable domain comprises theamino acid sequence shown in SEQ ID NO:
 2. 14. The cell of claim 9wherein the α chain variable domain comprises the amino acid sequenceshown in SEQ ID NO: 13 and the β chain variable domain comprises theamino acid sequence shown in SEQ ID NO:
 2. 15. The cell of claim 9wherein the α chain variable domain comprises the amino acid sequenceshown in SEQ ID NO: 12 and the β chain variable domain comprises theamino acid sequence shown in SEQ ID NO:
 15. 16. The cell of claim 9wherein the α chain variable domain comprises the amino acid sequenceshown in SEQ ID NO: 13 and the β chain variable domain comprises theamino acid sequence shown in SEQ ID NO:
 15. 17. The cell of claim 9wherein the α chain variable domain comprises the amino acid sequenceshown in SEQ ID NO: 12 and the β chain variable domain comprises theamino acid sequence shown in SEQ ID NO:
 14. 18. The cell of claim 9wherein the α chain variable domain comprises the amino acid sequenceshown in SEQ ID NO: 13 and the β chain variable domain comprises theamino acid sequence shown in SEQ ID NO:
 14. 19. A pharmaceuticalcomposition comprising a plurality of cells as claimed in any of claims3 to 18, together with a pharmaceutically acceptable carrier.